臺灣博碩士論文加值系統

目 錄
指導教授推薦書
口試委員會審定書
致謝 ------------------------------------------------------------------------iii
中文摘要 -------------------------------------------------------------------------iv
英文摘要 -------------------------------------------------------------------------vi
目錄 ------------------------------------------------------------------------vii
圖目錄 -------------------------------------------------------------------------x
表目錄-------------------------------------------------------------------------xi
第一章 序論 ----------------------------------------------------------------1
第一節 肝癌簡介 ----------------------------------------------------------1
第二節 肝癌的基因體研究 ----------------------------------------------2
第三節 癌症基因的篩選 -------------------------------------------------3
第四節 AMHR2 可能為抑癌基因 -------------------------------------3
第五節 AMH 的訊號傳遞路徑 -----------------------------------------5
第六節 研究動機與實驗架構 ------------------------------------------5

第二章 材料與方法 ----------------------------------------------------------7
第一節 細胞株及細胞培養 ----------------------------------------------7
第二節 細胞數目之測定 -------------------------------------------------7
第三節 組織檢體來源 ----------------------------------------------------8
第四節 基因表達質體構築 ----------------------------------------------8
第五節 細菌轉型作用 ----------------------------------------------------8
第六節 細菌培養及菌種保存 -------------------------------------------9
第七節 基因轉殖 --------------------------------------------------------10
第八節 質體DNA中量製備 --------------------------------------------11
第九節 細胞 RNA 萃取 ------------------------------------------------12
第十節 細胞 RNA 的反轉錄 ------------------------------------------ 12
第十一節 即時定量聚合酶鏈鎖反應 ----------------------------------13
第十二節 轉殖 shRNA 病毒 -------------------------------------------- 13
第十三節 蛋白質萃取和西方墨點法 ----------------------------------14
第十四節 細胞增生實驗 -------------------------------------------------15
第十五節 免疫組織化學染色 (Immunohistochemistry, IHC) ------16

第三章 實驗結果 ------------------------------------------------------------17
第一節 觀察 AMHR2 在肝癌臨床的表現量 -----------------------17
第二節 觀察 AMHR2 在肝癌臨床⽅方⾯面的意義 --------------18
第三節 觀察 AMHR2 在惡性程度不同之肝癌細胞中的表現量 -18
第四節 AMHR2 的質體構築 ------------------------------------------19
第五節 在肝癌細胞中，增加 AMHR2 的表現量 -----------------19
第六節 增加 AMHR2 的表現量，對肝癌細胞⽣長的影響 -----20
第七節 在肝癌細胞中，抑制 AMHR2 的表現量 -----------------21
第八節 在 AMHR2 表現的肝癌細胞株中加入 AMH 濃度測試-21
第九節 表現 AMH 蛋白質在培養液的情形 -------------------------22
第十節 在肝癌細胞中大量表現 AMH 蛋白質或 AMHR2 蛋白質時會增加 Smad 1 / 5 / 8 磷酸化蛋白質跟 p21 蛋白質的表現 -----------------------------------------------------------------22

第四章 討論 ------------------------------------------------------------------23
參考文獻 -------------------------------------------------------------------------48

附圖目錄
圖一、利用中研院 Lentivirus-based shRNA library 進行腫瘤形成能力的篩選過程-------------------------------------------------------------27
圖二、cDNA microarry 資料分析顯示 AMHR2 mRNA 的表現量與肝癌的分期相關性 -------------------------------------------------------28
圖三、在 GEO 資料庫結果顯示 AMHR2 在腫瘤表現量低於非腫瘤部分 -------------------------------------------------------------------------29
圖四、AMHR2 蛋白表現量在腫瘤組織中低於非腫瘤組織 ----------30
圖五、利用免疫組織染色的方式觀察 AMHR2 在腫瘤組織和非腫瘤組織中的表現量 -------------------------------------------------------31
圖六、AMHR2 在肝癌細胞株中的表現量 -------------------------------32
圖七、構築 AMHR2 帶有 Flag-tag 或綠螢光蛋白質體的流程圖 ----33
圖八、在 Huh7 細胞株中 AMHR2 表現分布在細胞質與細胞膜的位置 -------------------------------------------------------------------------35
圖九、肝癌細胞株中大量表現 AMHR2 時其 serine 位置有被磷酸化的現象產生 -------------------------------------------------------------36
圖十、 AMHR2 會影響 Smad 1 / 5 / 8 跟 p21 的訊息傳遞 ------------37
圖十一、AMHR2 的表現會抑制肝癌細胞株的生長 -------------------38
圖十二、利用 shRNA 抑制肝癌細胞株 AMHR2 基因表現的情況 --39
圖十三、 利用 siRNA 抑制肝癌細胞株 AMHR2 基因表現的情況 --40
圖十四、AMH 會活化 Smad 1/5 的訊號傳遞 ----------------------------41
圖十五、表現 AMH 蛋白質在培養液的情形 ----------------------------42
圖十六、在肝癌細胞中大量表現 AMH 蛋白質或 AMHR2 蛋白質時會增加 Smad 1 / 5 / 8 磷酸化蛋白質跟 p21 蛋白質的表現 -------------------------------------------------------------------------43
表一、分析 AMHR2 與臨床因子間的關係 -------------------------------44
表二、引子資訊 ----------------------------------------------------------------45
表三、抗體資訊 ----------------------------------------------------------------46
表四、 shRNA 和 siRNA 序列 ------------------------------------------------47

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